Human Molecular Genetics, 2002, Vol. 11, No. 23 2989-2996
© 2002 Oxford University Press
Up-regulation of c-Jun N-terminal kinase pathway in Friedreich's ataxia cells
1BioGeM Consortium, Federico II University, Naples, Italy, 2Department of Molecular and Cellular Biology and Pathology and IEOS, CNR, Federico II University, Naples, Italy and 3Department of Neurology, Federico II University, Naples, Italy
Received July 31, 2002; Accepted August 28, 2002
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ABSTRACT |
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The severe reduction in mRNA and protein levels of the mitochondrial protein frataxin, encoded by the X25 gene, causes Friedreich ataxia (FRDA), the most common form of recessive hereditary ataxia. Increasing evidence underlines the pathogenetic role of oxidative stress in this disease. We generated an in vitro cellular model of regulated human frataxin overexpression. We identified, by differential display technique, the mitogen activated protein kinase kinase 4 mRNA down regulation in frataxin overexpressing cells. We studied the stress kinases pathway in this cellular model and in fibroblasts from FRDA patients. Frataxin overexpression reduced c-Jun N-terminal kinase phosphorylation. Furthermore, exposure of FRDA fibroblasts to several forms of environmental stress caused an up regulation of phospho-JNK and phospho-c-Jun. To understand if this susceptibility results in cell death, we have investigated the involvement of caspases. A significantly higher activation of caspase-9 was observed in FRDA versus control fibroblasts after serum-withdrawal. Our findings suggest the presence, in FRDA patient cells, of a ‘hyperactive’ stress signaling pathway. The role of frataxin in FRDA pathogenesis could be explained, at least in part, by this hyperactivity.
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INTRODUCTION |
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Friedreich ataxia (FRDA) is the most common form of recessive hereditary ataxia (1), typically caused by a large GAA repeat expansion within the first intron of the X25 gene (2). Expanded GAA tracts have been shown to produce a severe reduction in mRNA and protein levels of the mitochondrial protein frataxin, encoded by the X25 gene (3–5). Patients develop ataxia, hypertrophic cardiomyopathy and, in a third of cases, diabetes or carbohydrate intolerance (1,6). The main pathological changes are the loss of large sensory neurons in dorsal root ganglia, followed by degeneration of posterior columns, spinocerebellar and pyramidal tracts (7,8).
Cardiomyocytes and pancreatic ß-cells have been suggested to be independent sites of primary degeneration (9). Increasing evidence underlines the pathogenetic role of oxidative stress in FRDA. Knock-out of the yeast frataxin homolog (YFH) produces mitochondrial accumulation of free iron, hypersensitivity to oxidative stress and depletion of mitochondrial iron–sulphur proteins (10–13). Also, the phenotypic features of the conditional frataxin KO mouse show mitochondrial iron overload and impairment of iron–sulphur proteins (14). The mitochondrial iron excess could lead to the formation of toxic oxygen radicals by Fenton reaction. An increased sensitivity to exogenous reactive oxygen species (ROS) has been demonstrated in FRDA fibroblasts (15) and lymphoblasts (16). An impairment of antioxidant enzymes has been demonstrated both in FRDA patients and in FRDA fibroblasts (17–19). Previous studies have reported that human frataxin overexpressing cells show decreased ROS levels and increased glutathione peroxidase activity and thiols (20). During retinoic acid-induced neurogenesis of frataxin deficient cells, an increase in apoptosis susceptibility has been shown, which was partially mediated by ROS (21). Recent evidence suggests that ROS may modulate a variety of signaling pathways, including the pathway of the c-Jun N-terminal kinase (JNK) (22). Our work is focused on the characterization of stress kinase pathways in FRDA.
We generated an in vitro model of regulated frataxin overexpression. In this model, by the differential display technique (23), we identified a decrease in the mitogen activated protein kinase kinase 4 (MKK4) transcript after frataxin overexpression. Starting from this observation, we investigated stress pathway activation in our cell models. Here, we report the involvement of the JNK pathway in the cell response to changes of frataxin levels.
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RESULTS |
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Regulated transgenic overexpression of human frataxin in PC12 rat cells
We generated an in vitro cellular model of regulated human frataxin overexpression (tet-off system), in which the increase in frataxin expression was caused by removal of the tetracycline analogue, doxycycline (dox). This model was used to identify mRNAs differentially expressed after frataxin overexpression. Rat pheochromocytoma cell line (PC12) was stably transfected with the plasmid pTRE-X25, containing the entire human frataxin coding sequence. Twelve clones were selected after transfection and analysed by western blot. Five clones resulted in a regulated overexpression of human frataxin upon dox withdrawal. One of those five human frataxin overexpressing clones (PL11) was selected. Immunoblot analysis of clone PL11 allows detection of human frataxin expression as early as 2 h after dox removal followed by a time-dependent increase, which reaches its maximal expression level between 8 and 24 h (Fig. 1). No evident modification of cell morphology resulted after exogenous human frataxin overexpression.
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Identification of decreased MKK4 transcript by differential display
To identify genes regulated by human frataxin overexpression, in transgenic cells, we used differential display technique (23). In this experiment, we compared total RNAs from clone PL11 in frataxin overexpression and basal conditions. We identified five differentially expressed transcripts. One of these transcripts, reduced by frataxin overexpression, was found to be the mitogen activated protein kinase kinase 4 (MKK4), by GeneBank sequence analysis. To confirm MKK4 mRNA down regulation in frataxin overexpressing cells we performed northern blot and real time RT–PCR experiments. As shown in Figure 2A, transcript levels of MKK4 were decreased up to
2.5-fold in frataxin overexpressing cells. To further confirm these data, we carried out a real time RT–PCR of RNAs isolated from two different cellular clones (PL11 and PL25) during a time course experiment of frataxin overexpression (Fig. 2B). MKK4 gene expression was down regulated during frataxin overexpression in both cellular clones. Already after 3 h we observed a 2-fold decrease of MKK4 mRNA that remained stable until 24 h of frataxin overexpression.
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Effect of human frataxin overexpression on the stress kinase pathway in transgenic cells
Because MKK4 is an activator of the c-Jun N-terminal kinase (JNK), also called stress-activated protein kinase (SAPK), we sought to examine the JNK pathway after exposure of the PL11 cellular clone to redox stress. We treated transgenic cells, before and after human frataxin overexpression, with 200 µM H2O2 and monitored changes in JNK phosphorylation. As shown in Figure 3, frataxin overexpression reduced JNK phosphorylation. At the 30 min time point, the level of phospho-JNK (P-JNK) was 3-fold lower in frataxin overexpressing cells compared to cells with a basal expression.
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Stress kinase pathway activation in fibroblasts from Friedreich's ataxia patients
To further investigate the role of frataxin expression on JNK activation we used primary fibroblasts from patients with the FRDA gene defect. In particular, we used fibroblasts derived from two adult patients (FRDA1 and FRDA2) with the clinical FRDA phenotype, and one from a foetus (FRDA3) with the FRDA gene defect. Frataxin levels, as expected, were found to be reduced in all three FRDA fibroblast cultures. All FRDA fibroblasts showed frataxin levels less than 15% of that of the controls. We examined the changes in JNK phosphorylation in three FRDA and three control (CNTL) fibroblasts, following exposure to 10 µM H2O2. As shown in Figure 4A, FRDA fibroblasts exhibited significantly greater JNK phosphorylation levels versus control fibroblasts. In particular, P-JNK in the FRDA cells increased up to 5-fold compared with controls after 10 min of H2O2 treatment. Similar differences, in time and concentration dependent patterns were observed in all FRDA–control pairs checked. No difference was observed between adult and foetal FRDA fibroblasts. To further explore JNK activation we performed serum-withdrawal experiments. In control fibroblasts, P-JNK increased after serum-withdrawal with a moderate peak at 24 h. In FRDA fibroblasts an earlier and higher peak was detectable at the 9 h time point (Fig. 4B). FRDA fibroblasts were further examined by investigating the regulation of a downstream component of the JNK pathway: c-Jun. We examined the changes in c-Jun phosphorylation following H2O2 treatment in FRDA and control fibroblasts. As shown in Figure 5, c-Jun phosphorylation was up regulated after H2O2 treatment in FRDA fibroblasts compared with control fibroblasts. Taken together, these data demonstrated that exposure of FRDA fibroblasts to environmental stress caused an up regulation of phospho-JNK and phospho-c-Jun.
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Caspase-9 activation in FRDA fibroblasts after serum-withdrawal
Our investigations supported the general idea that FRDA cells have a high susceptibility to stress conditions. To understand if this susceptibility results in cell death, we have investigated the involvement of the caspases, a family of cysteine proteases modulating the morphological changes characterizing the apoptotic process. Particularly, after a serum-withdrawal time-course we analysed the activation of caspase-9, the critical effector for the apoptotic stimuli acting through mitochondrial dysfunction. A significantly higher activation of caspase-9 was observed in FRDA versus control fibroblasts after serum-withdrawal (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The exact physiological function of frataxin is not yet known. Several predominant hypotheses for its role have been made: mitochondrial iron transport; iron–sulphur (Fe–S) cluster biogenesis; and response to oxidative stress. Several authors have suggested that frataxin is a protein able to keep iron in bioavailable and non-toxic form (24,25). These findings could explain the involvement of frataxin in so many diverse functions. Recently, other authors stated that it is still unclear whether frataxin is directly involved in iron binding (26). To investigate the function of frataxin we have generated a rat pheochromocytoma cell model overexpressing human frataxin in a regulated manner. In this model, human frataxin overexpression limits the activation of JNK after treatment with H2O2. JNK represents one subgroup of mitogen-activated proteins (MAP kinases). MAP kinase signaling pathways relay, amplify and integrate signals from a diverse range of extracellular stimuli, thereby controlling the physiological response of a cell to changes in the environment (27). JNK is activated primarily by exposure to environmental stress (e.g., redox stress, osmotic stress and radiation) (28). The JNK subgroup has also been known as stress-activated protein kinase. A major target of the JNK signaling pathway is the activation of AP-1 (activator protein-1) transcription factor that is mediated, in part, by the phosphorylation of c-Jun (29,30) (Fig. 7). Non-inducible overexpression of human frataxin in murine 3T3L1 cells has been previously reported (20,31). These cells exhibit both an increased resistance to exogenous oxidative stress and decreased endogenous levels of ROS. Our findings are consistent with these data. In light of the results presented here, it is tempting to speculate that the frataxin overexpression decreases levels of ROS, likely subtracting iron to Fenton reaction. This decrease of ROS might limit the JNK activation. The reduction of JNK activation may render these cells more resistant to oxidative stress. Frataxin overexpressing 3T3L1 cells also show elevation in oxidative phosphorylation (OXPHOS) and in mitochondrial capacity (31). It has been demonstrated that JNK inhibits the activity of the metabolic enzymes glycogen synthase kinase 3b (GSK-3) (32). The resulting change in GSK-3 activity leads to augmented glycogen synthase (GS) activity, with a subsequent increased conversion of glucose to glycogen. The ability of frataxin to limit JNK activation could explain the elevation of mitochondrial energy conversion and oxidative phosphorylation found in frataxin overexpressing 3T3L1 cells. Data obtained from FRDA fibroblasts confirmed the results obtained from regulated transgenic frataxin overexpressing cells. Both JNK and c-jun phosphorylation, after stress stimuli, are increased in FRDA fibroblasts in comparison to controls. Our findings suggest the presence, in FRDA patients cells, of a ‘hyperactive’ stress signaling pathway. It is noteworthy that this hyperactive stress signaling pathway seems to be present in fibroblasts both from patients with clinical phenotype and from a foetus with only the FRDA genetic defect. This would suggest that the disregulation of JNK phosphorylation in FRDA is a very early event in the pathogenesis of the disease. A deficiency of frataxin might favor iron participation in Fenton reaction that, in turn, can activate the stress signaling pathway. Cardiac manifestations are conspicuous in FRDA. About one-half of patients die of heart failure and nearly three-quarters have evidence of cardiac dysfunction throughout life (1,6). Almost all patients have an abnormal echocardiogram in the form of symmetric, concentric, hypertrophic cardiomyopathy. It is intriguing that previous reports demonstrated that the activation of JNK alone is sufficient to induce characteristic features of cardiac hypertrophy (33). It has been previously demonstrated that cells from FRDA patients have an increased susceptibility to being killed by oxidative stress (15,16). We observed in fibroblasts from patients, a strong activation of caspase-9 after 48 h of serum-withdrawal. In this condition very low activation is observed in control cells. JNK is required for the stress-induced release of mitochondrial cytochrome c (34) that, in turn, activates caspase-9. We suggest that the increased susceptibility of FRDA cells to die by oxidative stress is mediated by JNK. Santos et al. demonstrated that frataxin deficient embryonic cells show enhanced apoptosis and generate more ROS during neuronal differentiation (21). It is well known that the JNK-dependent apoptotic signaling pathway can be counterbalanced by the activation of survival signaling pathways (NF-
B, Akt/PKB and ERK) (34). It is possible to speculate that a ‘hyperactive’ stress signaling pathway renders frataxin-deficient embryonic cells less responsive to neurotrophins and affects their decision between survival and apoptosis. The loss of dorsal root ganglia (DRG) neurons, observed in FRDA patients, could be explained, at least in part, by this mechanism. In the apoptosis of the frataxin deficient embryonic cells Bcl-2 seems not to be involved since there was a comparable expression between frataxin deficient and control cells. It is intriguing that it has been demonstrated that Bcl-2 may not be the physiological substrate of JNK (29). The finding that JNK pathway activation is reduced in the frataxin overexpressing model (transgenic PC12) and increased in low expressing cells (FRDA fibroblasts) is an additional suggestion for a direct, likely via ROS, role of frataxin in JNK regulation. In summary, our results suggest that at least a part of the role of frataxin in Friedreich's ataxia pathogenesis is related to up regulation, probably via ROS, of the JNK stress pathway. The JNK signaling pathway has been implicated in many pathological conditions, including cancer, stroke, heart disease, inflammatory diseases and neurodegenerative diseases (30,31,35). Pharmacological strategies to block JNK activity could be of therapeutic value, considering the implication of the JNK signal transduction pathway in so many pathological conditions (29). It is possible that the JNK signaling pathway represents a potential target also for FRDA therapeutic intervention.
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MATERIALS AND METHODS |
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Cell lines
The PC12 Tet-off cell line was obtained from Clontech (Palo Alto, CA). PC12 Tet-off cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% FBS, 10% HS, 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). The entire coding region of human frataxin (X25) cDNA was EcoR1-inserted into the eukaryotic expression vector pTRE (Clontech). For stable transfection of 1x106 PC12 Tet-off cells, 12 µg of pTRE-X25 expression plasmid (containing G418 resistance) plus 1.5 µg of hygromycin resistance-conferring plasmid ( pTK-Hyg; Clontech) were co-transfected with lipofectAMINE Reagent (Invitrogen). For clonal selection, 200 µg/ml hygromycin (Roche, Mannheim, Germany), 100 µg/ml G418 and 10 ng/ml doxycycline (Sigma, Munich, Germany) were used. Primary fibroblast cultures were established from skin biopsies of two patients with classical FRDA and two normal, unaffected controls, following written informed consent. One primary fibroblast culture was derived from aborted foetus' (at 18 weeks of pregnancy) skin tissue, after molecular diagnosis of FRDA and another primary fibroblast culture was derived from skin tissue of a control foetus (at 18 weeks of pregnancy), following parents written informed consent. Analysis of GAA repeat expansion was performed by PCR as described (36) and the following allele sizes (number of triplets) were determinated: FRDA 1: 983/1093; FRDA 2: 527/634; FRDA 3 (foetus): 679/679; control 1: 9/8; control 2: 8/8; control 3 (foetus): 8/8. Fibroblasts were grown in DMEM supplemented with 2 mM L-glutamine, 20% FBS, 100 U/ml penicillin and streptomycin. Transgenic PC12 and fibroblasts were treated with 200 µM and 10 µM H2O2 respectively, and cells were harvested at the indicated time. Serum withdrawal from fibroblast cultures was obtained replacing fresh DMEM serum-free medium, after two washes in DMEM serum-free medium and cells were harvested at the indicated time. All experiments were conducted on fibroblasts between passages 7 and 11.
RNA extraction and cDNA synthesis
Total RNA was isolated from cells using ‘TRIZOL reagent’ according to the manufacturer's instructions (Invitrogen). 2 µg of total RNA was reverse transcribed with 100 U Super Script II Rnase H- Reverse Transcriptase (Invitrogen) in a volume of 40 µl, using 100 µM random hexamer primers (Roche) according to the manufacturer's instructions (Invitrogen).
Northern blot
15 µg of total RNA of the PC12 PL11 clone, grown in the absence of doxycycline (dox-) for 24 h and in the presence of doxycycline (dox+), were electrophoresed on a 1% denaturing formaldehyde agarose gel and transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech, Buckinghamshire, England). A 1250 bp MKK4 cDNA fragment, a 1020 bp glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA fragment and a 700 bp FRDA (X25) cDNA fragment were DIG-labelled by PCR reaction using DIG-dUTP (Roche) and used as probes. Hybridization and detection of mRNA was performed according to the manufacturer's instructions (Roche).
Differential display
The identification of differentially expressed RNAs in PC12 PL11 after 24 h of frataxin overexpression was performed using ‘Delta Differential Display Kit’ (Clontech). PCR products were electrophoresed through 5% polyacrylamide gels. The gels were then stained using the silver staining protocol (Promega, Madison, WI, USA). Differentially expressed bands were extracted, re-amplified, cloned in TOPO TA Cloning (Invitrogen) and sequenced. Sequence analyses were performed in GeneBank database.
Real-time quantitative PCR
A quantitative assay for rat MKK4 mRNA expression was estabilished using GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). All quantifications were normalized to an endogenous control using the 129 bp fragment at 3' of the rat beta-2-microglobulin region. PCR oligo-primers were: Rat-MKK4 forward primer 5'-GCAGCTGAGTAATTCTGAGGAAAGG-3' and Rat-MKK4 reverse primer 5'-CGGCACG TTCTTCATACATCAAAATAA-3' generating a 200 bp fragment; Rat-B2M forward primer 5'-GACCGATGTATATGC TTGCAGAGT-3' and Rat-B2M reverse primer 5'-GGATCT GGAGTTAAACTGGTCCAG-3'. Real-time PCR was performed using the SYBR Green PCR Master Mix 2X (Applied Biosystems) and 50 ng of cDNA in a total volume of 25 µl. Each sample was run in triplicate for both MKK4 and beta-2-microglobulin. PCR cycling profile consisted in AmpErase UNG Incubation for 2 min at 50°C and AmpliTaq Gold Activation for 10 min at 95°C as pre-denaturation steps and in 50 two-step cycles at 95°C for 15 seconds and at 60°C for 60 seconds. For MKK4 mRNA relative quantification was used the comparative Ct method according to the manufacturer's instructions (Applied Biosystems).
Western blot analysis
Transgenic PC12 cell lines and fibroblasts were lysed in a buffer containing 10 mM Tris ( pH 7.4), 150 mM NaCl, 1 mM EDTA ( pH 8.0), 1% Triton X-100, with phosphatase inhibitor (25 mM ß-glicerolophosphate, 2.5 mM sodium pyrophosphate) and protease inhibitor (Complete, EDTA-free protease inhibitor cocktail (Roche)). The fibroblast cell extracts for Caspase-9 immunoblot were prepared using Chaps Cell Extract Buffer (Cell Signaling, Beverly, MA, USA) according to the manufacturer's instructions. Total lysates were incubed for 30 min on ice and insoluble material was removed by centrifugation at 10 000 g at 4°C for 15 min. Protein concentration was measured using the Bio-Rad protein assay. Extracts were denatured and separated on 12% or 15% SDS-polyacrylamide gels, transferred to a Hybond ECL nitrocellulose membrane (Amersham) by electroblotting and blocked overnight with 10% non-fat milk. The following primary antibodies were used: anti-frataxin monoclonal antibody 1G2 (Chemicon International, Tehecula, CA, USA); anti-SAPK/JNK antibody, anti-phospho-SAPK/JNK antibody, anti-phospho-c-Jun antibody, anti-Caspase-9 antibody (Cell Signaling); anti-
-tubulin antibody (Sigma). Secondary antibodies (anti-mouse IgG or anti-rabbit IgG (Sigma)) coupled to peroxidase were used for detection of the reaction with the ECL kit (Amersham), according to the manufacturer's instructions. The protein bands were scanned and the intensity of each band was corrected according to the intensity of the -tubulin or JNK bands as indicated. The relative intensities were quantified using ‘Scion Image’ software.
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ACKNOWLEDGEMENTS |
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This work was supported in part by a grant from BioGeM Consortium.
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FOOTNOTES |
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* To whom correspondence should be addressed at: BioGeM s.c.a.r.l. c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università "Federico II", Via S. Pansini, 5, 80131 Napoli Italy. Tel: +39 0817462354; Fax: +39 0817463011; Email: pianese{at}biogem.it
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